Process for the preparation of immunogammaglobulin using trithionate compounds

ABSTRACT

A process for the preparation of immunogammaglobulin derivatives that can be administered by intravenous injection because of their reduced anticomplement activity level in which the reaction of human immunogammaglobulin with a compound capable of forming trithionate ion and another compound capable of forming sulfite ion in water is carried out in water to cleave the interchain disulfide bonds in said immunogammaglobulin and simultaneously the sulfur atoms cleaved are S-sulfonated.

TECHNICAL FIELD

The present invention relates to a process for the preparation ofimmunogammaglobulin. More particularly, the present invention relates toan improved process for the preparation of S-sulfonatedimmunogammaglobulin.

BACKGROUND ART

Immunoglobulin includes antibodies against a variety of diseases and isused for the prophylaxis and therapy of these diseases, however, the useof immunoglobulin preparation has been limited to intramuscularinjection. Namely, when an immunoglobulin preparation is intravenouslyinjected, the immunoglobulin binds with the complement to lower theserum complementary concentration and cause a side-effect calledanticomplementary action, resulting in the observation of blood pressuredrop, body temperature rise, disorder in circulatory system and others.Therefore, many studies have been made to attain the purpose of keepingimmunoglobulin stable on intravenous administration.

The first method is to cut off the part that is thought to bind with thecomplement from human immunoglobulin by use of enzyme. For example, A.Nisonoff proposed to use pepsin as an enzyme [c.f. Science 132. 1770(1970)] and another method using plasminogen in serum, cathepsin andothers as an enzyme is described in the specification of Japanese Pat.No. 47-37529 (1972). However, these enzyme-treated immunoglobulins havedefects that their half-life period is short and consequently theduration time of the efficacy is short as E. Merler and B. Jager showedin their articles of Vox Snag 13, 102 (1967) and Arch. Intora. Med. 119,60 (1967) respectively.

The second method is to treat human immunoglobulin with aprotein-acylating reagent. For example, Japanese Patent Laid-open49-6119 (1974) described that the acylation of human immunoglobulin gavea modified immunoglobulin with reduced anticomplement activity-level.But the acylated immunoglobulin formed by this process has a danger ofgiving rise to antigenecity in human bodies and the administration inlarge amounts has been thought to be difficult.

The third method is to reduce the disulfide bonds in humanimmunoglobulin, followed by alkylation. For example, according toJapanese Patent Laid-open No. 48-103723 (1973), this method gave amodified immunoglobulin having the same apparent molecular weight asthat of the unmodified immunoglobulin and a reduced anticomplementactivity level. However, this method is a two-stepped process and theoperations are considerably complicated, thus being industriallydisadvantageous.

The fourth method is to S-sulfonate the interchain disulfide bonds inhuman immunoglobulin with tetrathionate ion and sulfite ion to giveimmunoglobulin derivatives suitably used for intravenous injection [c.f.Japanese Patent Laid-open Nos. 50-121421 (1975), 51-1630, 51-76418, and51-112512 (1976)]. This method is the best in known ones. But, in thismethod unstable tetrathionate salt is used as an oxidant. Therefore, alarge amount of the tetrathionate salt is required to promote thecomplete reaction, which oftentimes causes side-reactions and furtherproduces the denaturation of the protein to inhibit the sufficientreduction in the anticomplement activity level as a defect to beimproved. Further, the oxidation power of tetrathionate ion isrelatively strong to have a possibility to break intrachain disulfidebonds as well as interchain disulfide bonds and sufficient caution isinconveniently required to control the reaction.

DISCLOSURE OF THE INVENTION

The present inventors focused their research of the improvement of suchdefects and have found that when trithionate ion, which has highstability and appropriate reactivity, and sulfite ion are together usedas oxidants, interchain disulfide bonds in human immunoglobulin arecleaved and simultaneously the cleaved sulfur atoms are S-sulfonated(--S--SO₃ ⁻) to prepare immunoglobulin derivatives that are suitablyused for intravenous injection because of freeness of the above defects,thus attaining the present invention.

Namely, the present invention is a process for the preparation ofimmunoglobulin derivatives that is characterized by conducting areaction of human immunoglobulin with a compound capable of producingtrithionate ion and another compound capable of producing sulfite ion inwater to cleave the interchain disulfide bonds in the humanimmunoglobulin and simultaneously to S-sulfonate (--S--SO₃ ⁻) thecleaved sulfur atoms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(a), (b) and (c) show patterns of electrophoretic migration ofimmunoglobulin (IG), sulfonated gammaglobulin (GGS) obtained in Example1 and another sulfonated gammaglobulin obtained in the reference ondodecyl sulfonate discs respectively. FIG. 1(d) shows the relationshipbetween each band in the above patterns and molecular weight.

FIGS. 2(a) and (b) show the gel filtration patterns of sulfonatedgammaglobulin (GGS) obtained in Example 1 and another sulfonatedgammaglobulin (GGS) obtained in the reference using Sephadex G 200respectively.

BEST MODE OF CARRYING OUT THE INVENTION

Any compound can be used as a trithionate ion resource, one of theoxidants used in the present invention, if it can form trithionate ionin water, however, an alkali metal salt of trithionate such as sodiumtrithionate or potassium trithionate is preferred.

Any compound can be used as a sulfite ion resource, if it can formsulfite ion in water, however, a preferred example is sulforous acid,sodium sulfite, sodium bisulfite, potassium sulfite or sodiumpyrobisulfite.

The amount of the compound capable of forming sulfite ion is twice ormore, preferably more than 10 times the molar quantity of the interchaindisulfide bonds to be cleaved in immunoglobulin. The amount of thecompound capable of forming trithionate ion is more than one mole,preferably two or more moles per mole of the interchain disulfide bondsto be cleaved in immunoglobulin. The reaction is effected in water andthe pH is preferably kept in the range from 6.0 to 10.0 during thereaction.

The reaction temperature is 50° C. or lower, preferably in the rangefrom 10° to 45° C. When the temperature exceeds 50° C., theimmunoglobulin molecule undesirably becomes more susceptible to proteindenaturation while temperatures lower than 10° C. extremely retard theprogress of the reaction, thus being industrially unpractical.

The reaction time, in which almost of the interchain disulfide bonds inimmunoglobulin are cleaved and S-sulfonated, depends upon the amount ofthe oxidants and the reaction temperature. In general it is chosen inthe range from 0.5 to 24 hours.

According to the present invention, immunoglobulin is cleaved in almostof the interchain disulfide bonds and the disulfide is converted toS-sulfonate groups (--S--SO₃ ⁻) to give the H-chain and L-chain. Thereaction product is separated by a usual purification method such asdialysis, salting-out or column chromatography. For example, thereaction mixture is dialyzed with normal saline solution to give theobjective substance in normal saline solution.

The present invention will be further illustrated by the hereinafterpresented examples. It is to be understood, however, that these examplesare presented as illustrative only and that is in no way intended tolimit the present invention thereto.

REFERENCE

1. Preparation of sodium trithionate

The objective compound was obtained from sodium thiosulfate and 30%aqueous hydrogen peroxide through a method described in new series ofexperimental chemistry ("Shin Jikken Kagaku Koza") Vol 8. Syntheses ofinorganic compounds (II), P482, Maruzen Tokyo.

Substantially no change was observed when it was stood at roomtemperature for several months. Further, the heating of the aquoussolution at 45° C. for 4.5 hours caused scarcely changes, too.

2. Preparation of sodium tetrathionate

Sodium tetrathionate was prepared from sodium thiosulfate and iodinethrough a method described in the Handbuch der PraparativenAnorganischen Chemie, Vol 1. p 362, Ferdinand Enke Verlag Stuttgart1960.

The product fell to 85% in purity when stood at room temperature for oneweek to evolve the odor of sulfur dioxide. Further, the heating of theaqueous solution at 45° C. for 4.5 hours resulted in the reduction ofpurity to lower than 50%.

EXAMPLE I

A human immunoglobulin 16% concentration solution in normal salinesolution that is buffered to 7.5 pH with phosphate solution (5 ml) wascombined with normal saline solution (5 ml) that contains sodium sulfite(0.1616 g., 6.4 times the molar quantity of the interchain disulfidebonds in immunoglobulin) and sodium trithionate (0.076 g., 16 times themolar amount of the interchain disulfide bonds) and is buffered to 7.5pH with phosphate solution and the reaction was effected at atemperature of 45° C. for 4.5 hours. After the completion of thereaction, the reaction mixture was dialyzed with normal saline solutionuntil the reactants became 0.1 mMol/l or lower in concentration toobtain 11 ml of sulfonated gammaglobulin 7% concentration solution innormal saline solution.

The anticomplement activity level CH₅₀ was determined on the 5% solutionin accordance with the method described by Kabat and Mayer (ExperimentalImmunochemistry, p 221, 1961) and found to be 14.9%.

The CH₅₀ of the 5% solution of human immunoglobulin, the startingmaterial, was 90%.

The titre of anti-diphtheria of the product was found to be 0.8units/ml, which was the same level as that of the human immunoglobulin,the starting material.

Further, the product was subjected to the sodium dodecylsulfonate-discelectrophoresis in accordance with a method by Weber and Osborne (J.Biol. Chem. 244, 446, 1969). The result is given as FIG. 1(b), whichdefinitely shows that the produce is almost composed to the H-Chain andL-chain and the interchain disulfide bonds have been sulfonated.

In the meantime, the same reaction was carried out using ³⁵ S-labelledsodium sulfite to confirm that 7-8.5 moles of --S--SO₃ ⁻ groups wereintroduced per mole of the immunoglobulin.

The pattern of immunoelectrophoresis was found to coincide entirely withthat of the product obtained by use of sodium tetrathionate [c.f. FIG.1(c)].

As a reference, the pattern of human immunogammaglobulin is given inFIG. 1(a).

Further, the gel filtration pattern using Sephadex G-200 is shown asFIG. 2(a). The OD₄₅₀ of 5% solution was found to be 0.950.

EXAMPLE II

The reaction was carried out in the same manner as in Example I exceptthat 0.015 g. (3.2 times the molar quantity of the interchain sulfidebonds) of sodium trithionate was employed to form sulfonatedgamma-globulin with anticomplement activity level CH₅₀ of 15.2%.

REFERENCE

A human immunoglobulin 16% concentration solution in phosphate-bufferednormal saline solution (5 ml) was combined with another normal salinesolution buffered to 7.5 pH with phosphate solution (5 ml) that containssodium sulfite (0.1616 g., 64 times the molar quantity of the interchaindisulfide bonds in the immunoglobulin) and sodium tetrathionatedihydrate (0.098 g., 16 times the molar quantity of the interchaindisulfide bonds) and the reaction was effected at a temperature of 45°C. for 4.5 hours. After the completion of the reaction, the reactionmixture was dialyzed with normal saline solution until the concentrationof the reactants became lower than 0.1 mMol/l to give 11 ml ofsulfonated gammaglobulin 7% concentration solution in normal salinesolution.

The anticomplement activity level CH₅ of the 5% solution was found to be20.2% and OD₄₅₀ was 0.112.

The pattern of the sodium dodecylsulfate disc electrophoresis of theproduct is given in FIG. 1(c) and the gel filtration pattern usingSephadex G-200 is shown in FIG. 2(b).

INDUSTRIAL APPLICABILITY

In the present invention, relatively stable trithionate ion and sulfiteion are used as sulfonating reagent and undersirable decomposition ofthese reagents can be avoided. Thus, large amounts of the reagentsbecome unnecessary and it leads to less side-reactions. Consequently,the present invention has advantages of giving immunogammaglobulinderivatives suitably used for intravenous injection because they havelow anticomplement activity level and turbidity and can be readilypurified by very simple operations.

The S-sulfonated immunogammaglobulin prepared according to the presentinvention returns to the original immunoglobulin in vivo and resists thedecomposition in blood with no danger of producing antigenecity.Moreover, said S-sulfonated immunogammaglobulin has characteristics ofenabling intravenous injection because of its reduced anticomplementactivity level without any adverse effect on various kinds of antibodyactivities and of having very long duration period of the efficacy invivo.

We claim:
 1. A process for the preparation of immunogammaglobulinderivatives in which the reaction of human immunogammaglobulin with acompound capable of forming trithionate ion and another compound capableof forming sulfite ion in water is carried out in water to cleaveinterchain disulfide bonds in said immunogammaglobulin andsimultaneously the sulfur atoms cleaved are S-sulfonated (--S--SO₃ ⁻).2. A process for the preparation of immunoglobulin derivatives accordingto claim 1 wherein the compound capable of forming trithionate ion inwater is one selected from sodium trithionate, potassium trithionate ortheir mixture.
 3. The process of claims 1 or 2, wherein said compoundcapable of forming trithionate ion is present in an amount greater than1 mol/mol of said disulfide bonds to be cleaved and said compoundcapable of forming sulfite ion is present in an amount of at least 2mol/mol of said interchain disulfide bonds to be cleaved.
 4. The processof claim 3, wherein said trithionate ion forming compound is present inan amount of 2 or more mols/mol of said interchain disulfide bonds to becleaved, and said sulfite ion forming compound is present in an amountexceeding 10 mols/mol interchain disulfide bond to be cleaved.
 5. Theprocess of claims 1 or 2, wherein the pH of the reaction mixture ismaintained in the range of from 6.0-10.0.
 6. The process of claims 1 or2, wherein the reaction temperature is maintained at 50° C. or less. 7.The process of claims 1 or 2, wherein the reaction temperature ismaintained in the range of 10° C.-45° C.
 8. The process of claim 4,wherein the pH of the reaction mixtures maintained at 6.0-10.0 and thereaction temperature is maintained in the range of from 10° C.-45° C.